1. Field of the Invention
The present invention relates to an analyte evaluation apparatus which has an analyte space for holding a liquid containing an analyte that includes a protein or the like (hereunder the liquid being sometimes called an analyte solution), a working electrode, a counter electrode and a reference electrode, and in which the potential of the working electrode is altered with respect to the potential of the reference electrode as the standard potential (that is, the potential difference between the working electrode and reference electrode is altered with respect to the potential of the reference electrode as the standard potential), and the behavior of the analyte is observed to thereby evaluate the analyte. When the analyte includes a protein or the like, this apparatus is simply called a voltage-driven protein sensor or voltage-driven protein chip. Consequently, the analyte evaluation apparatus of the embodiments in this specification can sometimes be called a voltage-driven analyte sensor or voltage-driven analyte chip.
2. Description of the Related Art
Voltage-driven protein chips are of interest because they can detect a wide variety of proteins rapidly and with high sensitivity, and since they are usually small enough to be held in the palm of the hand, they are easily applicable to medical settings and inspections (see for example Yoshinobu BABA, “Nihon no biokei μ-TAS saishin gijutsu” (Biological μ-TAS latest technologies in Japan), Bioindustry Tokushugo (exclusive reports), 2007, Vol. 24(2), p. 5-78).
The typical structure of a voltage-driven protein chip can be explained for example as follows. A nucleotide probe, which is a nucleotide having bound thereto a target protein and a fluorescent dye or other fluorescent label, is fixed via a thiol group for example to a working electrode made of a metal gold film for example. The nucleotide probe is usually negatively charged, and applying a rectangular wave potential (for example, ±200 mV) to the working electrode causes the nucleotide probe to stand up from the working electrode by electrostatic repulsion or lie down on the working electrode by electrostatic attraction.
This movement is called the switching movement of the nucleotide probe. If the nucleotide probe is exposed to excitation light from an argon ion laser for example to excite the fluorescent label when the nucleotide probe is standing and elongated, fluorescent light is emitted, while almost no light is emitted (that is, the probe is quenched) if the fluorescent label is excited in the same way when the nucleotide probe is lying down and/or contracted. The fluctuation range of the fluorescent intensity due to repeated lighting (that is emission) and quenching of the fluorescent label is called the fluorescent intensity switching amplitude.
If the cycle of the potential applied to the working electrode is as low as 0.5 Hz, the switching movement of the nucleotide probe will be synchronized with the cycle of the potential. However, if the cycle is as high as 1 kHz or higher for example, the switching movement of the nucleotide probe will not be able to follow the cycle of the potential, and the fluorescent intensity switching amplitude will decline, while the rising and falling parts of the rectangular waveform will become more gentle. This change in the waveform is called decreased frequency responsiveness. Moreover, when the nucleotide probe is bound to a protein the mass is much greater than the mass of the nucleotide probe without the protein (10 times more for example), causing the switching movement of the nucleotide probe no longer to be able to follow the cycle of the potential, resulting in a decrease in fluorescent intensity switching amplitude and decreased frequency responsiveness. Furthermore, because of the short distance (a few to 100 nm for example) between the fluorescent dye and the protein bound to the nucleotide probe, the protein absorbs (quenches) the fluorescence, also causing a decrease in fluorescent intensity switching amplitude.
In a voltage-driven protein chip, it is possible to determine the presence or absence, or type of a target protein bound to the nucleotide probe or to measure its concentration with high sensitivity based on such decreases in fluorescent intensity switching amplitude and frequency responsiveness. Although a voltage-driven protein chip was explained above, the above explanation can be applied to a voltage-driven analyte chip or to any analyte evaluation apparatus described in this specification. In this case, the element including the fluorescent label and responding part with the analyte can be seen as corresponding to the nucleotide probe described above. An element including a fluorescent part and a responding part with an analyte in this way is sometimes simply called a probe hereunder.
In the embodiments described in this specification, “evaluating an analyte” means determining the presence or absence, type and/or concentration of an analyte as discussed above, and an “analyte” is a substance to be evaluated in this way, such as a protein for example, which may or may not include parts having the function of enabling evaluation, such as the aforementioned fluorescent label or the aforementioned part that may be standing and/or elongated, or lying down and/or contracted (hereunder sometimes called the responding part) or parts having the function of binding to the working electrode (such as a thiol group). When the analyte does not include these elements, they are added to the analyte or to the working electrode at certain stages up to evaluation.
A counter electrode and reference electrode are necessary for applying voltage to the working electrode. In a voltage-driven analyte chip, the probability at which the analyte collides with the working electrode is much greater (about 100 times greater for example) if these electrodes are incorporated into a small channel (such as about 0.5 mm in height, 2.5 mm in width, 50 mm in length) than in a system in which the solution is simply agitated in a 50 mL beaker for example, allowing for more rapid evaluation.
However, in such a voltage-driven analyte chip bubbles may occur in the analyte solution as it flows through the channel, and when these bubbles pass over the electrodes or are present between electrodes, they may impede electrical conduction between the working electrode and counter electrode or between the working electrode and reference electrode, making it difficult to accurately apply the desired potential difference between the working electrode and reference electrode. As a result, not only can the behavior of the analyte not be detected accurately, but if an excessively large potential difference is applied the probe may become desorbed from the working electrode or become oxidized, and there will be no switching movement signal to be observed among other problems. These bubbles are believed to occur due to pressure changes and/or temperature changes in the channel, which reduce the solubility of gasses in the analyte solution in the channel. This problem was explained with reference to an analyte solution flowing in a channel, but may occur even when the analyte solution is retained in the analyte space rather than flowing in a channel.
When conduction between electrodes is broken due to bubbles, one possible means of dealing with the problem is to break the application of voltage in the circuit, but this is not easy for the following reasons. For example, the applied voltage needs to be broken before ion rearrangement occurs around the electrodes, but even if the applied voltage is greater than ±500 mV, if the circuit is not broken within a few hundred nanoseconds with overshoot controlled within 100 mV, the probe may become desorbed from the working electrode or the probe adsorbed on the working electrode may become oxidized so there is no switching signal to observe. However, such a circuit is not easy to achieve.
Another possible method of preventing bubbles would be to first degas the liquid with a degassing unit before supplying it to the channel, but it would still be difficult to completely prevent bubbling.
Bubbles are less likely to occur in a liquid if the liquid is made to flow in the channel by application of pressure rather than suction, but leakage from the channel then becomes more likely, and the structure of the unit is complicated by the measures taken to prevent leakage, detracting from productivity when the apparatus is produced.